1. Field of the Invention
The present invention relates to a method of detecting a nucleotide sequence. The present invention also relates to a method of analyzing a nucleotide, in particular a method of analyzing a gene.
2. Description of the Related Art
A method of detecting nucleotide mutation by using specificity of a ligase enzyme was reported in Science in 1988 (Landegren, Science, 26; 241 (4869): 1077-80, (1988)). The method is much easier in controlling the reaction condition than the traditional mutation detection method by PCR, i.e., the mutation detection method by PCR using a primer having a mutation-complementary sequence at the 3′ terminal (i.e., Sequence Specific Primer). For this reason, various more advanced methods such as LCR (ligase chain reaction) of using a heat-resistant ligase in the subsequent step and LDR (ligase detection reaction) by Barany et al. were developed since then (Jpn. Pat. Appln. KOKAI Publication No. 2000-511060). Molecule recognition with ligase is performed in combination of two probes having a nucleotide complementary to mutation at the 3′ terminal. In particular, the method has been used frequently for detection of a single nucleotide polymorphism.
Human SNP (single nucleotide polymorphism) is a gene polymorphism occurring at a frequency of approximately one out of hundreds of bases. The mutation occurs over a wide range of genome, independently of the coding or non-coding region, in the form of substitution, insertion, or deletion of bases. The size of the human genome is 3 billion base pairs, and a frequency of 1/1,000 base means presence of 3,000,000 SNPs. It is not easy to identify a medically useful particular SNP out of the vast number of SNPs. The number of SNP sets sensitive to a medicine or disease is thought to be about hundreds or dozens in the approximately 3,000,000 SNPs. For example, Roche provides a SNP-typing microarray for a medicine-metabolizing protein cytochrome P450. The array is designed to type a total of 31 alleles: 29 alleles for gene CYP2D6 and 2 alleles for gene CYP2C19. Thus, it seems that there is no need for typing tens or hundreds of thousands alleles for diagnosis. Practically, SNP typing of dozens, at most a hundred and several tens, of alleles would be needed.
However, even in typing of such a degree, use of the conventional Sanger's method results in increase in cost for the reagents and apparatus for reaction and also lower detecting efficiency of one mutation in one reaction. In addition, when the reaction specificity or signal is lower, it is not always possible to read the SNP from the waveform obtained. Under the circumstances above, the number of the samples to be detected is too many for the Sanger's method, in continuing typing as described above. Examples of the other typing methods include SSCP (single strand conformation polymorphism) method simpler in experimental procedure, SSP-PCR (sequence specific primers-PCR) method, real time PCR method by using a fluorescent TaqMan probe, and the like. However, these methods are also yet to be commercialized. Thus, there is a need for a method allowing analysis of many SNP types at lower cost, for commercialization of SNP testing. Currently, studies on the advanced methods, in particular based on the method of using the molecule recognition of polymerase or ligase, are eagerly in progress all over the world.
For example, a unique method by using molecule recognition of ligase is MIP (molecular inversion probe) method of Affymetrix (Jpn. Pat. Appln. KOKAI Publication No. 2004-528016). The method is a multiplex typing method of using a tag, which reduced the cost for probe synthesis and raised the reaction efficiency with a closed-ring probe and a gap ligation method (Hardenbol, P. et al., Nat. Biotechnol. 21, 673-678 (2003), Hardenbol, P. et al., Genome Res. 15, 269-675 (2005)). Alternatively, the RCA (rolling circle amplification) method developed by Lizardi of Yale Univ., an attractive nucleotide amplification method replacing PCR, is a method of producing a cyclic DNA from a primer with a strand-displacing polymerase continuously (Jpn. Pat. Appln. KOKAI Publication No. 2001-519172). The template for the amplification method should be cyclic. Thus, a padlock probe in which a terminal of single-strand chain probe hybridizes to a target and the hybrid is ring-closed by a ligase was developed, and a patent application on a detection method using the same was filed (Jpn. Pat. Appln. KOKAI Publication No. 2002-503948). Aisin Cosmos filed a patent application on a padlock probe method of using a protein RecA forming a triple-stranded chain and accelerating specific hybridization (Jpn. Pat. Appln. KOKAI Publication No. 9-220099) in Japan. In addition, the padlock probe is used not only in the RCA method but also in other methods (Jpn. Pat. Appln. KOKAI Publication No. 2001-514483, Japanese Patent Nos. 3085409 and 3590633).
In detecting mutation of a genome nucleotide with the padlock probe, a probe nucleotide is added in a great excess amount to a nucleotide sample amplified, for example by PCR or a reaction solution containing genome nucleotide itself for ligation reaction. It is necessary to prepare a detection probe, in addition to PCR primers, to perform such a reaction. Addition of a great excess amount of the probe nucleotide easily leads to increase of non-specific reaction. Further, when a nucleotide has a secondary structure in the region close to the detection sequence, the padlock probe hardly hybridizes to the object, leading to possible prohibition of detection.
By any one of the conventional methods above, for example when a nucleotide is detected by ligation reaction of the probe, a great excess amount of probe nucleotide should be added to the nucleotide sample, which leads to increase in cost and also increase of nonspecific reaction.